The mechanism of several phosphotransferases will be explored by the use of kinetic and chemical probes to detect and explore the properties of ionic and covalent enzyme-substrate intermediates. In addition to initial rate and equilibrium isotope exchange studies, we plan to initiate oxygen-18 scrambling experiments to detect intermediates which might not be observable by more standard protocols. The interaction of regulatory molecules with these phosphotransferases will be examined to determine the particular steps in the catalytic process that are modulated allosterically. Moreover, the interactions of cyclic AMP at the regulatory subunit of protein kinase will be explored to map the topology of the kinase substrate and regulatory sites. The kinetic theory that will permit an increased understanding of cooperative systems will be developed by studies on a dimeric acetate kinase from an anaerobe.